Haynes Lab:Notebook/Jan/2015/04/08: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 21: Line 21:
* Calculations
* Calculations
** Vector - MV2(E/X); '''50 ng''' = '''1.5 μL'''
** Vector - MV2(E/X); '''50 ng''' = '''1.5 μL'''
** Insert - BD004(E/S); (4096/4529)*2*50 = '''90.4 ng''' = '''### μL'''
** Insert - BD004(E/S); (4096/4529)*2*50 = '''90.4 ng''' = '''2.8 μL'''




Line 27: Line 27:
# BD004 + MV2
# BD004 + MV2
# MV2 only (neg)
# MV2 only (neg)
# BD004 only (neg)- to check if any of old vector co-purified w/ insert; bands were very close on gel




Line 34: Line 35:
| bgcolor=#cfcfcf | 1
| bgcolor=#cfcfcf | 1
| bgcolor=#cfcfcf | 2
| bgcolor=#cfcfcf | 2
| bgcolor=#cfcfcf | 3
|-
|-
| DNA insert || 1.4 || ---  
| DNA insert || 2.8 || --- || 2.8
|-
|-
| DNA vector || 7.7 || 7.7
| DNA vector || 1.5 || 1.5 || ---
|-
|-
| 2x buffer || 9.6 || 9.6
| 2x buffer || 5.0 || 5.0 || 5.0
|-
|-
| T4 ligase || 0.5 || 0.5  
| T4 ligase || 0.5 || 0.5 || 0.5
|-
|-
| dH<sub>2</sub>O || --- || 1.4
| dH<sub>2</sub>O || 0.2 || 3.0 || 1.7
|-
|-
| &nbsp; || 19.2 μL  || 19.2 μL   
| &nbsp; || 10.0 μL  || 10.0 μL || 10.0 μL   
|}
|}


Revision as of 11:48, 8 April 2015

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

04/08/15

  • Assembly - BD004/MV2


Assembly


  • Calculations
    • Vector - MV2(E/X); 50 ng = 1.5 μL
    • Insert - BD004(E/S); (4096/4529)*2*50 = 90.4 ng = 2.8 μL


  • Ligations
  1. BD004 + MV2
  2. MV2 only (neg)
  3. BD004 only (neg)- to check if any of old vector co-purified w/ insert; bands were very close on gel


Reagent 1 2 3
DNA insert 2.8 --- 2.8
DNA vector 1.5 1.5 ---
2x buffer 5.0 5.0 5.0
T4 ligase 0.5 0.5 0.5
dH2O 0.2 3.0 1.7
  10.0 μL 10.0 μL 10.0 μL

--> RT/ ~30 min.

  • Add 50 μL DH5-α turbo
  • 5 min./ ice
  • Plate on 100 ug/mL amp agar. Grow at 37 °C o/n


RESULTS

  • After ~7 hours of growth, saw tiny colonies only on ligation plate
  • To save time, picked two colonies from ligation plate
    • Make streaks on fresh agar
    • Used tips to inoculate 5 mL cultures


  • 4/07/15
    • Success! ~20 colonies in ligation plate, zero on negative ctrl.
    • Growth on streak plate and in cultures



Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Jan/2015/04/08&oldid=878869"